Since the advent of recombinant DNA, methods have been developed and improved for the purification of DNA and RNA to further molecular biology research. While these methods have allowed considerable study of nucleic acids in research environments, methods for preparative scale production of plasmid DNA sufficient in quantity and quality for clinical use have been problematic and continue to represent an unmet need.
Gene therapy involves the introduction of nucleic acid into a patient's cells, which, when expressed, provide a therapeutic benefit to the patient. Examples include the introduction of an exogenous, functional gene to correct a genetic defect in a patient carrying a defective gene or to compensate for a gene that is not expressed at sufficient levels. Other examples include the introduction of mutant genes, antisense sequences or ribozymes to block a genetic function, e.g., in the treatment of viral infections or cancer.
For any application in which nucleic acid is introduced into a human or animal in a therapeutic context, there is a need to produce highly purified, pharmaceutical grade nucleic acid. Such purified nucleic acid must meet drug quality standards of safety, potency and efficacy. In addition, it is desirable to have a scaleable process that can be used to produce multiple gram quantities of DNA. Thus, it is desirable to have a process for producing highly pure nucleic acid that does not require toxic chemicals, known mutagens, organic solvents, or other reagents that would compromise the safety or efficacy of the resulting nucleic acid, or make scale-up difficult or impractical. It is also desirable to prepare nucleic acids free from contaminating endotoxins, which if administered to a patient could elicit a toxic response. Removal of contaminating endotoxins is particularly important where plasmid or bacteriophage DNA is purified from gram-negative bacterial sources that have high levels of endotoxins as an integral component of the outer cell membrane.
Preparative scale plasmid manufacturing most commonly involves alkaline lysis of bacterial cells containing extrachromosomal DNA of interest such as plasmid or phage DNA. Alkaline lysis was first developed by Birnboim and Doly, Nucleic Acids Res 1979; 7(6):1513-23, as a screening method for recombinant plasmids. As Birnboim and Doly found, alkaline lysis of bacteria effects release of intracellular plasmid DNA together with selective denaturation of chromosomal DNA that renatures upon neutralization to form an insoluble “clot” together with cellular debris. The lysate from an alkaline lysis process, such as for example the method of Birnboim and Doly, usually consists of a slurry of precipitated or flocculated debris suspended in a golden yellow colored liquid. The plasmid DNA remains predominantly in the liquid portion of the neutralized solution. To obtain the liquid containing desired extrachromosomal polynucleotides, the debris has to be removed from the slurry without shearing of either the precipitated chromosomal DNA debris or the extrachromosomal polynucleotide products. As in the minipreparative technique of Birnboim and Doly, centrifugation is the most commonly used method to isolate the liquid from the solid precipitates. On a preparative scale, various means have been employed to clarify the alkaline lysate including centrifugation, sedimentation, and filtration. Centrifugation has been commonly employed. (Bussey et al., U.S. Pat. No. 6,011,148; Butler et al., U.S. Pat. No. 6,313,285). Filtration means have included bag filtration (Thatcher et al., U.S. Pat. No. 5,981,735), depth filtration (Mittelstaedt and Hsu, U.S. Pat. No. 6,268,492) and filtration with diatomaceous earth (Horn et al., U.S. Pat. No. 5,576,196).
While continuous-flow centrifugation can be efficient for the separation, the shearing motion can result in irreversible damage to the plasmid. On the other hand, batch centrifugation involves complete containment of the lysate inside centrifuge bottles, which prevents the degradation during centrifugation. However, scaling up the batch centrifugation process has been constrained by the lack of commercial availability of larger centrifuges. Also, having multiple centrifuges may not be feasible, due to the prohibitive expense. Thus, as the scale of the production run increases, the volume of material makes the traditional centrifugation, sedimentation, and filtration means too inefficient, time consuming and/or expensive. A more efficient method of removing the precipitated debris is therefore needed.
The invention described provides a novel method and apparatus for clarification of cell lysates that is rapid, highly efficient, relatively inexpensive and conducive to automation.